Abstract Objective To explore protein levels in oral squamous cell carcinoma tumor suppressor gene PTEN expression and clinical pathological significance. Methods SP immunohistochemical staining of tissue microarray technology to detect 10 cases of normal oral mucosa, 10 cases of oral epithelial hyperplasia, 15 cases of oral leukoplakia and 72 cases the OSCC (including 30 cases of well-differentiated, differentiated, 26 cases, poorly differentiated 16 cases) of PTEN protein expression, analysis of the relationship between PTEN expression with clinical and pathological data. Results of the PTEN gene in oral squamous cell carcinoma, oral leukoplakia, oral epithelial hyperplasia and normal oral mucosal tissue, the positive rates were 72.2% (52/72), 93.3% (14/15), 100% (10/10) oral squamous cell carcinoma and other groups, a significant difference (P <0.05) and 100% (10/10); positive expression rate of PTEN in different gender, age and TNM staging clinicopathological parameters no significant 0.05），但与淋巴结转移、组织分化程度等临床病理参数间有显著性差异（P<0.05）。">difference (P> 0.05), but with lymph node metastasis, histological grade and clinical pathological parameters significant difference (P <0.05). Conclusion PTEN gene expression down in one of the OSCC occurrence and development plays an important role in the expression of abnormal reference can be used as prognostic indicators.
Key words】 the PTEN; oral squamous cell carcinoma; tissue microarray; immunohistochemistry
Oral squamous cell carcinoma (Oral squamous cell carcinoma, OSCC) is the most common malignant tumors of the oral and maxillofacial region, and its mechanism is not yet clear. Mutations and abnormal expression of oncogenes and tumor suppressor genes plays an important role in the process of tumor formation. Newly discovered tumor suppressor gene PTEN is the first phosphatase activity of tumor suppressor genes, and the occurrence and development is closely related to a variety of malignancies, downregulation promote tumor development, but the the PTEN and OSCC relationship has not been reported. We are using tissue microarray and immunohistochemical detection PTEN expression in OSCC, and to explore the role of PTEN occur in OSCC development and transfer.
1 Materials and methods
1.1 The object of study
1999 to 2004, 72 cases of oral squamous cell carcinoma surgery archived paraffin-embedded specimens collection of Luzhou Medical College Department of Pathology, and randomly selected 10 cases of normal oral mucosa, 10 cases of oral epithelial hyperplasia and paraffin-embedded specimens of 15 patients with oral leukoplakia. All the cases were the first onset, had not received preoperative radiotherapy and chemotherapy. Patients 49 male patients and 23 females; aged between 22 to 86 years old, with a median age of 58 years old. Which lymph node metastasis in 37 cases, 35 cases without lymph node metastasis. All specimens were 10% neutral formalin-fixed, paraffin-embedded, 4μm serial sections.
1.2 Reagents and Methods
1.2.1 Preparation of tissue microarray using handmade chips. (1) Positioning: The filter consists of two high qualification pathologist to identify representative tumor area, and make a mark on the corresponding paraffin blocks. (2) making the microarray wax block: first make a blank the receptor wax block. Embedded box system with a diameter of 1.8mm, a pitch of 2 mm, the transverse No. 1 to 7, the longitudinal direction A ~ F, a total of 42 orifices the blank receptor wax block. Then with an inner diameter of 1.7mm empty needles turn drill on the mark good wax block organizations turn into blank the receptor wax block. Will build the good arrays wax block into the mold and then placed in secondary embedded instrument the second embedded system out microarray paraffin blocks. (3) sliced, mounted sheet: to make good tissue microarray wax block to re-slice thickness is 4 μm. The sections were mounted onto poly-L-lysine treatment slide made of tissue microarray. Total production microarray containing 123 tissue samples, 16 replicate samples.
1.2.2 Immunohistochemical staining streptomycin avidin – peroxidase mildew connection method (SP method), that is, with mouse anti-human monoclonal PTEN antibody, SP kit, DAB chromogenic reagent were purchased from Beijing Zhongshan. Strictly according to kit instructions staining, all sections were stained under the same conditions. Instead of primary antibody with PBS as negative control, to do positive control with known positive prostate tissue sections.
1.3 The results of judgment
All immunohistochemical staining results by two pathologists blinded reading in case I do not know the clinical data judge observed under high magnification (× 400) for each slice at least 10 vision to judge the results of non-repetition. Positive parts of PTEN protein expression in the nucleus. Reference the Salvesen standard semi-quantitative method staining intensity was divided into four: No coloring; yellow 1; brownish yellow; tawny 3. The proportion of positive cells divided into four: the number of positive cells <10% 0; 10% to 40% for 1 minute; 40% to 70% for 2 minutes; ≥ 70% 3. The two ratings sum, 0 to 1 is divided into (-); 2 is divided into the (+); 3 to 4 is divided into the (+); 5 to 6 (+ + +). Statistical when (-) is negative, and the remainder is positive.
1.4 statistical methods
Application SPSS11.5 statistical software for the analysis of experimental data rate compared using the χ2 test.
2.1 tissue microarray quality wax block the process of preparation array unorganized core loss, tissue microarray arranged in neat rows. In the preparation of the chip during the 4 tissue folding, but does not affect the observation, no tissue loss. Immunohistochemical staining process without tissue loss.
2.2 Immunohistochemical staining results (1) the expression of PTEN protein in oral squamous cell carcinoma: the positive parts of the PTEN protein staining in the nucleus, brownish yellow granules. Positive expression in squamous cell carcinoma cells is the the cancer nests organization well differentiated cells or cancer beads around the cell, as shown in Figure 1. Normal oral mucosal epithelial hyperplasia of PTEN protein expression were positive, as shown in Figure 2; 15 patients with oral leukoplakia PTEN protein expression was 93.3% (14/15); PTEN protein expression in 72 patients with oral squamous cell carcinoma was 72.2 72% (52/72). Normal oral mucosa, oral epithelial hyperplasia the oral leukoplakia organization of PTEN protein expression and oral squamous cell carcinoma was statistically significant (P <0.05), as shown in Table 1. (2) the relationship between PTEN expression and clinical pathological parameters after χ2 test, PTEN expression in different gender, age and the TNM points during were not statistically significant; PTEN protein expression in high, medium, poorly differentiated tissue 0.05）；中分化组与">rate was 90% (27/30), 76.9% (20/26), 31.3% (5/16), was not statistically significant (P> 0.05) between the group of well-differentiated and moderately differentiated group; differentiated group 0.05）；高分化、中分化组合并后与低分化组之间有统计学意义（P<0.05），同时PTEN的表达与淋巴结转移之间也有">between poorly differentiated group not statistically significant (P> 0.05); well-differentiated and differentiated portfolio and poorly differentiated groups was statistically significant (P <0.05), while PTEN expression and lymph node metastasis statistically significant. PTEN protein expression significantly correlated with the histological grade and lymph node metastasis of OSCC, as shown in Table 2. Table 1 different organizations PTEN protein expression
3.1 Application of tissue microarray significance
The tissue microarray, also known as tissue microarrays (Tissue microarray) is a method of application of gene chip concept evolved. With a slice on a one-time completion of hundreds of cases of tissue gene amplification, mRNA transcription and gene expression analysis of the product . In this study, we constructed a large sample OSCC tissue microarray immunohistochemistry test results consistent findings reported in the literature using the traditional method. Us build the chip with oral squamous cell carcinoma good representation. Organization chip has the following advantages: ① The one ordinary slice contains a lot of tissue, the large amount of information, you can get a large amount of data, operating more standardization and comparability of results more convincing. (2) due to the production of tissue microarray drilled smaller organizations will not impact the specimen as a whole, and can repeatedly drill significant savings pathological resources, is an efficient, fast, low consumption. (3) can be used in many fields, such as research, teaching, and biological reagents testing. Of course, tissue microarray also has some limitations, is As we derived relatively small, not entirely representative of conventional chipper. However, our study is a whole rather than the individual, so not much impact on the results. Moch  organizational differences the of tissue microarray ordinary slice contrast study found that: The results obtained in the ordinary slices can get on a tissue microarray. In most cases, the test results of the tissue microarray is able to represent the organization.
3.2 PTEN protein expression in oral squamous cell carcinoma, the development of the relationship between
PTEN (Phosphatase and homology to tensin homolog deleted on chromosome 10) gene is a tumor suppressor gene, discovered in 1997, it is targeted at 10q23.3, containing nine exons, encodes a protein of 403 amino acid-containing, distributed in the cytoplasm . PTEN is a highly conserved gene, plays an important role in cell life. Shin et al  of the entire sequence of the PTEN gene in 81 patients with microsatellite stable and 5 patients with microsatellite unstable OSCC detection, found that there were four cases of PTEN gene missense mutation, two cases of the OSCC chromosome 5 exon sub missing. Cairns  that the PTEN gene loss rate is high, and the mutation rate is low, and therefore possible for the wild-type but not mutant PTEN protein detected by immunohistochemistry. Without nonsense mutations and missense mutations can make wt PTEN inactivation, leading to loss of protein expression. Somatic mutations or deletions inactivated when the PTEN gene, resulting in increased intracellular PIP3 levels, Akt overactive cells added value to the formation of tumors . Therefore, PTEN gene plays an important role in tumorigenesis and development process.
This experiment normal oral mucosa and hyperplasia tissue PTEN protein positive rate was 100% involved in the maintenance of oral epithelial cells of normal function, the regulation of normal epithelial cells of the material metabolism, cell proliferation and differentiation; prompt PTEN gene PTEN in oral leukoplakia positive expression rate was 93.3%, indicating abnormal function of the the oral leukoplakia organization has emerged; OSCC tissue PTEN was expressed in 72.2%, and normal oral mucosa, oral epithelial hyperplasia and oral leukoplakia tissues of PTEN expression differences statistically significant, which is similar to the findings of Lee et al  showed that PTEN plays an important role in the development of OSCC occurrence. Missing or weakened can not antagonize the role of protein kinase PI3K PTEN protein expression, the loss of a negative regulator of PI3K/PKB/AKT signal transduction pathway leading to abnormal function of the signal transduction pathways, which may make oral epithelial cells lose their normal cell cycle regulation, allowing the cells to proliferate indefinitely and form tumors. The role of PTEN in development of OSCC The occurrence in pending further study.
The experiment also showed that PTEN protein expression was high, moderately differentiated OSCC tissue PTEN protein expression was significantly higher than the poorly differentiated OSCC organization, the difference was statistically significant. PTEN protein expression level is lower, the higher the degree of malignancy of the tumor, suggesting that the gene plays an important role in the process of differentiation induction of tumor. This is consistent with findings in Squarize . In addition, the study also found that PTEN protein expression without lymph node metastasis was significantly lower than the expression rate of lymph node metastasis, the difference was statistically significant. This may be because the PTEN protein focal adhesion kinase (FAK) and Shc have a regulatory role, can inhibit tumor cell invasion and metastasis. This experiment PTEN protein staining in the nucleus, there are a few in the cytoplasm, and Wang Ying, etc.  reported that PTEN protein staining in the cytoplasm opposite. The exact mechanism needs further research.